Lignin is one of the most abundant polymers on earth, second only to cellulose. Its complex structure makes it highly resistant to microbial degradation. Consequently, lignin is the primary cause of recalcitrance of lignocellulosic feedstock, and the primary constituent of waste effluent from second-generation biofuel fermentation. The United States can generate 1.3 billion dry tons of lignocellulosic biomass annually without competing with food crops for land use, and hence potentially deliver an equivalent supply of 3.8 billion barrels of oils that can replace more than 50% of liquid transportation derived from fossil fuels. However, one major limitation is that lignocellulosic residuals (i.e., lignins) constituting about 30% of the total biomass content cannot be currently used for fermentation and are underused as a low-value heating source by biorefinery processes. Therefore, it is significant to develop enabling technologies for transformation of this underused biomass source into high-value chemicals, biofuels, and biomaterials.
Utilization of the effluent lignocellulose waste stream would improve the overall process efficiency of second-generation biofuel production because the additional product would offset operating costs. This would effectively decrease the cost of the ethanol or butanol products, making them more competitive with traditional fossil fuels. Valorization of this waste stream will decrease the cost of treatment for any producing industries. So research paradigms or commercial ventures need not retool their foundational goals or core business models to incorporate this process.
Second-generation biofuels are a renewable energy source produced from lignocellulosic biomass, and they are fully compatible with existing infrastructure. Biofuels are produced in large bioreactors using single-celled microorganisms to convert the biomass into ethanol, butanol, or other hydrocarbons via fermentation processes. These single-celled organisms are incapable of degrading lignin, and consequently, the lignocellulosic biomass is never fully converted into desired products. In addition, the lignin present in the biomass feedstock shields the cellulose and hemicellulose that the microorganisms utilize effectively preventing optimum yields even when lignin degradation is not considered. Thus, up to 30% dry weight of the feedstock remain as lignin-containing residuals and wastes after biofuel production. Beside biofuel productions, other industrial activities that use lignocellulosic feedstock (e.g., production of pulp or paper) produce important amounts of lignocellulosic wastes. The resulting lignin-enriched waste stream is toxic to many microbes and plants, which leads to complications in its disposal since it is considered as hazardous waste. For twenty years, main treatment of lignocellulosic waste consisted of burning such wastes or burying, both of which have huge impacts on the environment. Then interest for valorizing these wastes rapidly expended over the recent years, using them as combustible heating source, for conversion by pyrolysis into char, gas and oil and used in building composite material. However, all these treatments convert only up to 3% of the remaining lignin.
The current slate of demonstrated lignin-derived products is very small and limited to native carbon storage compounds and intermediates of aromatic catabolism. To increase the portfolio of products that can be made from lignin, other parts of metabolism will need to be targeted.
The TCA cycle is a source of many value-added chemicals including succinate and citrate, but it has not yet been harnessed for lignin valorization. Itaconic acid (and its salt, itaconate, which are used interchangeably herein) and trans-aconitic acid (and its salt, trans-aconitate, which are used interchangeably herein) are unsaturated dicarboxylic acids derived from the TCA cycle with industrial uses including as an acrylate alternative and for the production of plastics, latex and other polymers (da Cruz et al., 3 Biotech 8.3 (2018): 138). Itaconate has been produced from simple sugars since the 1950s (Kuenz, A. et al., Applied Microbiology, and Biotech. 102.9 (2018): 3901-3914), and its potential to functionally replace several petroleum-derived commodity chemicals was highlighted by its selection as one of the top bio-based platform chemicals in several reports, including a 2004 United States Department of Energy report (Werpy, T. et al, No. DOE/GO-102004-1992. National Renewable Energy Lab, Golden, Colo. (US), 2004). However, the high cost of sugars makes itaconate production expensive, limiting it to use as a specialty chemical. Using lignin, a cheap and abundant feedstock, for production would enable much broader industrial use of itaconate.
The saprophytic bacterium Pseudomonas putida KT2440 is a microbe of industrial interest due to its robust metabolism (Ebert, Birgitta E., et al., Appl. Environ. Microbiol. 77.18 (2011): 6597-6605) and tolerance to xenobiotics (Kieboom, J. et al., Journal of Biological Chemistry 273.1 (1998), 85-91; Fernández, M. et al., Microbial biotechnology 2.2 (2009): 287-294.; Inoue, A. et al., Nature 338.6212 (1989): 264). P. putida also has the ability to tolerate and catabolize a wide-range of aromatic compounds (Jiménez, J I. et al., Environmental microbiology 4.12 (2002): 824-841) which led to its recent use in upgrading depolymerized lignin into PHAs (Gong, T. et al., Microbial biotechnology 9.6 (2016): 792-800; Linger, Jeffrey G., et al., Metabolic engineering communications 3 (2016): 24-29) and cis, cis-muconic acid (Kohlstedt, M. et al., Metabolic engineering 47 (2018): 279-293; Linger, J G., et al., PNAS 111.33 (2014): 12013-12018). In P. putida, lignin-derived aromatics are funneled into the β-ketoadipate pathway, producing acetyl-CoA and succinate (FIG. 1A). This direct route to key TCA cycle intermediates suggests that high yields of TCA cycle-derived products such as itaconate should be possible from lignin.
Growth phase production of itaconate may be challenging because itaconate can disrupt bacterial growth via inhibition of enzymes in the glyoxylate shunt and citramalate cycle. An alternate approach is to use a two-stage process to decouple growth of the microbial catalyst from conversion of feedstocks to chemicals, which provides solutions to many problems present in growth-associated processes (e.g. product toxicity, slow catalyst growth) (Burg, Jonathan M., et al., Curr. Op. in Chem. Eng., 14 (2016): 121-136). Such processes often take advantage of the natural responses of microbes to various nutrient limitations (e.g., nitrogen, sulfur, phosphate) and environmental shifts (e.g., O2 limitation, temperature shifts) that prevent microbial growth while maintaining the metabolic reactions of interest and can be coupled with dynamic metabolic control tools to entirely reroute metabolism.
While itaconate is a valuable biologically-derived platform chemical, it inhibits the growth of many bacteria—particularly during growth on C1-C3 compounds—by inhibiting isocitrate lysate (Michelucci, Alessandro, et al., PNAS, 110.19 (2013): 7820-7825), which has limited industrial production to a few fungal species with narrow substrate ranges (Kuenz, A. et al., App. Microbio. & Biotech., 102.9 (2018): 3901-3914; da Cruz, Juliana Cunha et al., Biotech 8.3 (2018): 138). The use of Pseudomonas putida as a platform for itaconate production would broaden the range of industrially-relevant feedstocks that could be upgraded to include lignocellulosic hydrolysates, lignin streams (Rodriguez et al. Acs Sustain Chem Eng 5, 8171-8180 (2017); Linger, J G., et al., PNAS, 111.33 (2014): 12013-12018), pyrolysis oil (Jayakody, L N., et al., Energy & Environ. Sci., 11.6 (2018): 1625-1638.), and more.